Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis

Abstract

Background Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition.ResultsA total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 106.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally.ConclusionsThese data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Evaluation of Safety and Protective Efficacy of a waaJ and spiC Double Deletion Korean Epidemic Strain of Salmonella enterica Serovar Gallinarum

“…It has been described that some LPS deficient mutants ( waaJ, waaL, rfaH, rfbH, rfbG, et al .) were used in Salmonella DIVA vaccine research ( 22 , 29 , 30 , 34 , 38 ). It was reported that immunization of piglets with the Δ waaJ or Δ waaL mutants of S. enterica ser.…”

“…Enteritidis and S. enterica ser. Pullorum S06004Δ spiC Δ waaL were also reported to have DIVA features ( 22 , 34 ). Our results showed that the antibodies produced by SG005Δ waaJ and SG005Δ waaJ Δ spiC did not react with S. enterica ser.…”

“…The stronger is the hydrophobicity of the cell surface, the higher is the auto-aggregation ability of the cell. The auto-aggregation assay was performed based on a previously described method with some modifications ( 22 ). Briefly, SG005, SG005Δ waaJ , SG005Δ spiC , and SG005Δ waaJ Δ spiC were statically grown in 5 ml of LB broth at 37°C for 24 h in 15-ml conical tubes.…”

Pathoadaptive Alteration of Salmonella Biofilm Formation in Response to the Gallbladder Environment

“…3c). Mutations in rfbG have been shown to cause a rough colony morphology and loss of LPS O antigen (32). During a screen for Salmonella genes required for long-term systemic infection after intraperitoneal infection, mutants in O-antigen biosynthesis were negatively selected (33).…”

Safety and protective efficacy of Salmonella Pullorum spiC and rfaH deletion rough mutant as a live attenuated DIVA vaccine candidate

“…These results were compared with wild type S. Pullorum S06004. The in vitro growth characteristics analysis of the mutant and wild-type strains was performed by measuring the optical density (OD 600 ) of each strain cultured in 20 mL of LB broth at 37°C with shaking at 180 rpm as previously described ( Jiao et al., 2017 ). The OD 600 was determined every hour and the monitoring was continued for 16 h. The LPS rough phenotype of the S06004 ΔspiCΔrfaH was identified by slide agglutination test using O 9 monoclonal antibody (O 9 MAb) developed in our laboratory and acriflavine (Sigma-Aldrich, MO, St. Louis, MO) agglutination test ( Jiao et al., 2018 ).…”